Preparation method and application of immobilized beads of deodorizing bacteria

Abstract

The present invention discloses a preparation method and application of an immobilized pellet of deodorizing bacteria. The present invention has the following beneficial effects: (1) the present invention adopts raw materials such as polyvinyl alcohol, boric acid, sodium alginate, and calcium chloride to prepare the immobilized pellet of Enterococcus hirae IDO5, and the required equipment is simple and the preparation process is simple; (2) the immobilized pellet prepared by the present invention enlarges the contact area between the deodorizing microorganism Enterococcus hirae IDO5 and odorous substances, enhances the microorganism's resistance to the influence of adverse environment, and improves the adsorption and degradation efficiency of odorous substances; (3) the immobilized pellet of Enterococcus hirae IDO5 has a good deodorizing effect, with a degradation rate of indole in pig manure wastewater as high as 93.0%, and a degradation rate of skatole as high as 81.6%. It is applied to livestock and poultry breeding manure and breeding wastewater to effectively reduce the concentration of odorous substances such as indole and skatole, and can effectively reduce the odor pollution caused by livestock and poultry breeding.

Description

Technical Field

[0001] The invention relates to the technical field of environmental protection, in particular to a preparation method of immobilized small balls of deodorizing bacteria and application thereof. Background Art

[0002] With the large-scale and intensive development of modern livestock and poultry farming, farms have produced a large amount of livestock and poultry manure. If it is discharged on site without treatment, it will form non-point source pollution around the city. In addition, the odor pollution caused by livestock and poultry manure affects the health of farmed animals and surrounding residents. Therefore, how to minimize the pollution of livestock and poultry farming odor to the environment has become a problem that must be faced and solved in the sustainable development of the breeding industry.

[0003] Odor emissions from livestock and poultry farms mainly come from livestock and poultry houses, manure storage sites, composting rooms and sewage pools. The main odor components of odor substances in livestock and poultry manure are ammonia, sulfide, volatile fatty acids, indole and skatole (scientific name 3-methylindole). Among them, indole and skatole are the main odor pollutants in livestock and poultry manure. They are metabolites of nitrogen-containing compounds such as protein and amino acids fermented by hindgut microorganisms. They are typical nitrogen pollutants with a very strong fecal odor. The environmental pollution and public health problems caused by them have always been the focus of attention in the breeding industry. Therefore, how to reduce odor pollution such as indole and skatole emitted by the breeding industry is a technical problem that needs to be solved urgently in the livestock and poultry breeding process.

[0004] The microbial deodorization method, which is currently studied more, uses microorganisms to biodegrade and transform odor substances. It has the characteristics of low cost and environmental protection, and is an ideal method for removing odor. However, in practical applications, due to the more complex composition of pollutants, free bacteria show the characteristics of low concentration of effective degradation bacteria per unit volume, poor activity, weak competition with indigenous bacteria, and poor resistance to toxic invasion. Since the 20th century, many scientists have begun to study the use of microbial immobilization technology and apply it to wastewater treatment. So far, fixed microbial technology has become a common way of wastewater treatment. After immobilization, microbial cells have the advantages of being difficult to lose cells, anti-pollution, improved ability to degrade pollutants, and tolerance to adverse living environments. Immobilized microbial methods can generally be divided into two categories: physical immobilization and chemical immobilization. Physical immobilization methods mainly include adsorption method, embedding method, etc., and chemical immobilization methods include covalent bonding method and cross-linking method. In actual microbial cell immobilization, since each single immobilization method has its own shortcomings, a composite method is often used to immobilize microbial cells. Composite fixation can combine the advantages of a single immobilization method and avoid its shortcomings, so it is widely used in cell immobilization.

[0005] The applicant previously screened out a deodorizing strain that can degrade odor substances such as indole and skatole from pig manure samples degraded by black soldier fly larvae. The strain was identified as Enterococcus hirae and named Enterococcus hirae IDO5. However, in actual applications, the composition of aquaculture manure and aquaculture wastewater is complex. In order to improve the activity of Enterococcus hirae IDO5 in aquaculture manure and wastewater, enhance the resistance of microorganisms to adverse environmental influences, and improve the degradation efficiency of odor substances, it is necessary to prepare Enterococcus hirae IDO5 into economical and efficient immobilized pellets to meet the targeted needs of the treatment of odor pollution caused by aquaculture manure, aquaculture wastewater, etc., and to achieve effective treatment of odorous gases generated by aquaculture waste. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention provides a method for preparing immobilized pellets of deodorizing bacteria and applications thereof, which can effectively reduce odor pollution caused by livestock and poultry breeding.

[0007] In order to solve the above technical problems, the present invention provides the following technical solution: a method for preparing immobilized beads of deodorizing bacteria, comprising the following steps:

[0008] S1: prepare culture medium;

[0009] S2: using the culture medium prepared in step S1 to prepare an Enterococcus hirae IDO5 bacterial suspension;

[0010] S3: polyvinyl alcohol, sodium alginate and distilled water are mixed, heated in a water bath to form a PVA-SA embedding agent solution, and further sterilized at high temperature and high pressure and then cooled;

[0011] S4: adding the Enterococcus hirae IDO5 bacterial suspension prepared in step S2 to the PVA-SA embedding agent solution cooled in step S3, and stirring well;

[0012] S5: dissolving boric acid and calcium chloride in distilled water to prepare a H3BO4-CaCl2 crosslinking agent solution;

[0013] S6: The mixed solution prepared in step S4 is taken and added to the H3BO4-CaCl2 cross-linking agent solution in step S5 to obtain immobilized beads after cross-linking.

[0014] Preferably, in step S1, the culture medium is specifically LB culture medium.

[0015] Preferably, step S1 specifically comprises: dissolving peptone, yeast powder and sodium chloride in distilled water, adjusting the pH of the culture medium to 7.3, and sterilizing for 20 min.

[0016] Preferably, step S2 specifically comprises inoculating the Enterococcus hirae IDO5 strain into a sterilized LB medium.

[0017] Preferably, in step S3, the PVA-SA embedding agent solution is sterilized at high temperature and high pressure and then cooled to room temperature.

[0018] Preferably, in step S6, the mixed solution prepared in step S4 is sucked by a 2.5 mL syringe without a needle, and is slowly added dropwise to the H3BO4-CaCl2 cross-linking agent solution in step S5 at a uniform speed.

[0019] Preferably, in step S6, the cross-linking time of the mixed solution and the H3BO4-CaCl2 cross-linking agent solution is 2 hours.

[0020] Preferably, after step S6, the method further comprises step S7: washing the immobilized beads obtained in step S6 with distilled water and placing them in a refrigerator for standby use.

[0021] Preferably, in step S7, the immobilized beads are washed three times with distilled water and placed in a refrigerator at 4°C for standby use.

[0022] In order to solve the above technical problems, the present invention provides another technical solution as follows: application of immobilized pellets of deodorizing bacteria, wherein the immobilized pellets prepared by the above preparation method are applied to the treatment of aquaculture manure or aquaculture wastewater.

[0023] Compared with the prior art, the present invention provides a preparation method and application of immobilized beads of deodorizing bacteria, which have the following beneficial effects: (1) The present invention adopts raw materials such as polyvinyl alcohol, boric acid, sodium alginate, and calcium chloride to prepare immobilized beads of Enterococcus hirae IDO5, and the required equipment is simple and the preparation process is simple; (2) The immobilized beads prepared by the present invention expand the contact area between the deodorizing microorganism Enterococcus hirae IDO5 and the odorous substances, enhance the microorganism's resistance to the influence of adverse environment, and improve the adsorption and degradation efficiency of odorous substances; (3) The immobilized beads of Enterococcus hirae IDO5 have a good deodorizing effect, with a degradation rate of indole in pig manure wastewater as high as 93.0%, and a degradation rate of skatole as high as 81.6%. When applied to livestock and poultry manure and aquaculture wastewater, it can effectively reduce the concentration of odorous substances such as indole and skatole, and can effectively reduce the odor pollution caused by livestock and poultry breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a graph showing the effect of different boric acid dosages on indole degradation rates in the present invention;

[0025] Figure 2 The figure is the effect of the concentration of CaCl2 solution on the degradation rate of indole of the present invention;

[0026] Figure 3 The figure is a comparison of the appearance of the immobilized beads at different SA dosages of the present invention;

[0027] Figure 4 This is a graph showing the effect of SA dosage on indole degradation rate of the present invention;

[0028] Figure 5 The figure is a comparison of the appearance of the immobilized beads under different PVA dosages of the present invention;

[0029] Figure 6 This is a graph showing the effect of the amount of PVA used on the degradation rate of indole in the present invention;

[0030] Figure 7 This is a comparison diagram of the effects of different factors on the degradation rate of indole in the present invention;

[0031] Figure 8 The present invention is a flowchart of the steps of preparing the immobilized deodorizing bacteria pellets. DETAILED DESCRIPTION

[0032] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] The relevant experimental materials of the present invention: disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O), ammonium sulfate ((NH4)2SO4), potassium dihydrogen phosphate (KH2PO4), ferric chloride (FeCl3), peptone, yeast powder, sodium chloride (NaCl), polyvinyl alcohol (PVA, 72000), sodium alginate (SA), boric acid (H3BO4), calcium chloride (CaCl2), etc. are all analytically pure.

[0034] The relevant specific operation methods of the present invention are as follows:

[0035] (1) Preparation of culture medium:

[0036] Mineral salt medium (MSM): Dissolve Na2HPO4·12H2O 3.28 g / L, (NH4)2SO4 2.0 g / L, KH2PO4 2.0 g / L, and FeCl3 0.00025 g / L in distilled water and sterilize for 20 min.

[0037] LB medium: Dissolve 10.0 g / L peptone, 5.0 g / L yeast powder, and 10.0 g / L NaCl in distilled water, adjust the pH of the medium to 7.3, and sterilize for 20 min, corresponding to the above step S1.

[0038] (2) Preparation of Enterococcus hirae IDO5 bacterial suspension:

[0039] The Enterococcus hirae IDO5 described in the present invention is a deodorizing strain that can degrade odor substances such as indole and skatole, which was screened out from a pig manure sample degraded by black soldier fly larvae. The Enterococcus hirae IDO5 of the present invention has been deposited in the Guangdong Microbial Culture Collection Center (GDMCC) on March 28, 2024. The deposit address is 5th Floor, Dayuan Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou. The deposit number is GDMCC NO.64457, and the classification name is: Enterococcus hirae ( Enterococcus hirae )IDO5.

[0040] The above step S2 specifically includes inoculating the Enterococcus hirae IDO5 strain into 100 mL of sterilized LB medium at 37 o C, 170 r / min to the logarithmic growth phase, wash the bacteria with saline, and then use saline to prepare a bacterial suspension to make the OD of the bacterial suspension 600 =0.24.

[0041] (3) Preparation method of immobilized beads of Enterococcus hirae IDO5:

[0042] Take a certain amount of PVA and SA into a conical flask, add 100 mL of distilled water to make up the volume, heat and stir in a constant temperature water bath to fully dissolve, prepare PVA-SA embedding solution, and sterilize it by high temperature and high pressure. After the PVA-SA embedding solution cools to room temperature, add 10 mL of Enterococcus hirae IDO5 bacterial suspension (OD 600 =0.24) (the volume ratio of Enterococcus hirae IDO5 suspension to PVA-SA embedding agent solution is 1:10), and stir well. Use a 2.5 mL syringe without a needle to draw the mixture of the Enterococcus hirae IDO5 suspension and PVA-SA embedding agent solution, and slowly drop it into the H3BO4-CaCl2 crosslinking agent solution (a certain amount of H3BO4 and CaCl2 are dissolved in distilled water). After crosslinking for 2 hours, the immobilized beads are obtained, washed three times with distilled water, and placed in a 4°C refrigerator for use.

[0043] In order to determine the optimal ratio of the immobilized material, the present invention selects different H3BO4 dosages, CaCl2 solution concentrations, PVA and SA dosages as factors, and takes the performance of the immobilized beads and the degradation rate of indole by the immobilized beads as indicators to determine the optimal level of each factor and optimize the immobilization conditions; the percentages involved are all mass percentages.

[0044] ①The influence of H3BO4 dosage

[0045] The amount of PVA was controlled at 7.0%, the amount of SA was 1.0%, and the amount of CaCl2 was 3.0%. The effect of the amount of boric acid H3BO4 on the degradation of indole by the immobilized strain IDO5 (i.e., the above-mentioned immobilized beads) was studied. The amount of boric acid was used in the following gradient: 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%. The experimental results are shown in Tables 1 and Figure 1 .

[0046] ②Influence of CaCl2 solution concentration

[0047] The amount of PVA was controlled at 7.0%, SA at 1.0%, and boric acid at 4.0%, and the effect of CaCl2 dosage on the degradation of indole by immobilized strain IDO5 was studied. The concentration gradient of CaCl2 solution was set to 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, and 4.0%.

[0048] ③Influence of PVA dosage

[0049] The effect of PVA dosage on the degradation of indole by immobilized strain IDO5 was studied by controlling the dosage of SA at 1.0%, CaCl2 at 3.0%, and boric acid at 4.0%. The PVA dosage gradient was set to 6.0%, 7.0%, 8.0%, 9.0%, 10.0%, 11.0%, and 12.0%.

[0050] ④Influence of SA dosage

[0051] The amount of PVA was controlled at 7.0%, the amount of CaCl2 was 3.0%, and the amount of boric acid was 4.0%, and the effect of SA dosage on the degradation of indole by the immobilized strain IDO5 was studied. The SA dosage gradient was set to 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, and 3.5%.

[0052] After designing the above single factor test, prepare the immobilized beads according to the above steps. Compare the size, shape, color, adhesion, etc. of the immobilized beads prepared under different conditions under the same conditions to determine the performance of the immobilized beads. Select 5 mL of PVA-SA embedding agent solution to prepare the immobilized beads into 5 ml of MSM inorganic salt culture medium, and add 50 μL of 10 mg / ml indole (final concentration of the reaction system 100 mg / L) to detect the indole degradation effect of the immobilized beads. Reaction conditions: speed 0r / min, temperature 37 oC, pH = 9, reaction for 24 h. All experiments were repeated 3 times. Take 10 mL of methanol to 5 mL of indole reaction system, shake and mix thoroughly, filter the mixture through a 0.22 μm organic filter membrane and inject it into the injection bottle. The concentration of indole was detected by high performance liquid chromatography-photoelectric reactor-fluorescence detector (HPLC-PHR-FLD). Chromatographic conditions: the chromatographic column is XBridgeTM C18 (4.6 mm×250 mm, 5 μm), the mobile phase is acetonitrile: water = 60:40 (V / V), the flow rate is 1 mL / min, and the column temperature is 35 o C, elution time 15 min, fluorescence excitation wavelength 270 nm, emission wavelength 350 nm.

[0053] Single factor experiment results:

[0054] ① Effect of Boric Acid Dosage

[0055] The performance of the immobilized beads under different boric acid dosages is shown in Table 1 below. According to Table 1, when the boric acid dosage is too low, the shape of the beads is slightly irregular. As the amount of boric acid increases, the shape of the immobilized beads gradually becomes a regular circle, and the time for the beads to change from transparent to milky white is shorter, and the transparency gradually increases. In terms of adhesion, as the amount of boric acid increases, the adhesion of the beads increases, and they separate after stirring. As the concentration of boric acid increases, the adhesion of the beads increases. This may be because when the two beads are close to each other, the hydroxyl groups in PVA that have not reacted in time combine with each other under the action of high concentration of boric acid, and the two beads move closer under the action of force. However, after a period of time, the surface cross-linking of each immobilized sphere is basically completed, the properties tend to be stable, and the beads disperse under external stirring.

[0056] Table 1 Dropping phenomenon at different boric acid dosages

[0057]

[0058] like Figure 1 As shown in the figure, with the increase of boric acid (H3BO4) dosage, the degradation rate of indole within 24 hours increased with the increase of boric acid dosage. When the boric acid content was 1.5%, the indole degradation rate was as low as 67.3%, and when the boric acid concentration reached the highest saturation concentration of 4%, the indole degradation rate reached the highest 83.5%.

[0059] Combined with the morphology of the immobilized beads and their degradation rate of indole when using different boric acid dosages, it was concluded that the optimal dosage of boric acid in the process of immobilizing the beads was 4%, at which time the 24-hour indole degradation rate was 83.5%.

[0060] ②Influence of CaCl2 solution concentration

[0061] The phenomena when different CaCl2 solution concentrations are added are shown in Table 2 below. As can be seen from Table 2, when the calcium chloride concentration is too low, the shape of the immobilized spheres will be difficult to form, slightly irregular, and will produce adhesion. As the concentration increases, the spheres are regular circles, the shape is more regular, the size tends to be uniform, no longer changes significantly, and there is no adhesion. Moreover, they can quickly change from transparent to milky white, and the degree of transparency decreases with the increase of calcium chloride concentration. This is mainly because under the condition of higher concentration of CaCl2, after PVA and SA undergo acetalization reaction, Ca 2+ Replace Na + The linear polymer molecules are cross-linked to form a network structure, thereby stabilizing the properties of the small balls.

[0062] Table 2 The phenomenon of adding CaCl2 solution with different concentrations

[0063]

[0064] Depend on Figure 2 It can be seen that when the concentration of CaCl2 solution is in the range of 1.0%-3.0%, the indole degradation rate increases continuously between 47% and 83%, and then with the increase of the concentration of CaCl2 solution, the indole degradation rate decreases continuously.

[0065] Taking into account the performance of the immobilized beads and other factors, the optimal concentration of CaCl2 solution was 3.0%, at which the indole degradation rate was 83% within 24 h.

[0066] ③ SA dosage effect

[0067] As can be seen from Table 3 below, when the concentration of SA is too low, the mixed solution has good fluidity and is easy to drip, but the immobilized beads cannot be formed. As the SA concentration increases, the viscosity of the mixed solution increases. In the range of 1.0%-2.0%, the beads are uniform in size and are regular spheres, but when the SA concentration exceeds 2.5%, the mixed solution is too viscous and difficult to drip, and the beads begin to tail. When the SA concentration is 3.5%, they cannot be formed and immobilized beads cannot be obtained. The addition of SA to PVA immobilized beads increases the viscosity of the PVA-SA mixed solution and enhances the sphering effect. This is because the PVA flexible macromolecules and the SA containing a large number of ring structures are combined to form an interpenetrating network structure, which changes the entangled structure of the gel formed by the association of hydroxyl hydrogen bonds between the PVA macromolecular chains. The appearance of the immobilized beads at different SA dosages is shown in the figure. Figure 3 shown.

[0068] Table 3 Dropping phenomenon at different sodium alginate dosages

[0069]

[0070] In addition, by Figure 4 It can be seen that with the increase of SA dosage, the indole degradation rate increases, and the maximum can reach 92.8%. However, when the SA content is above 2.5%, with the increase of SA content, the shape of the immobilized beads becomes irregular and tailing occurs, so the increase in the indole degradation rate is due to the enhanced adsorption of indole by the irregular beads.

[0071] Therefore, taking all factors into consideration, when the SA dosage is 2%, the shape of the pellets is regular spherical without tailing and the degradation ability is high. Therefore, the optimal SA dosage is 2%, at which time the degradation rate of immobilized pellets indole is 82.8%.

[0072] ④Influence of PVA dosage

[0073] As shown in Table 4 below, when the PVA content is too low, the viscosity of the mixed solution is low and it is easy to add dropwise, but the immobilized beads will stick together. When the PVA content is too high (more than 10%), the mixed solution is too viscous and difficult to add dropwise. The higher the PVA concentration, the more serious the tailing of the beads. The appearance of beads with different PVA contents is as follows: Figure 5 shown.

[0074] Table 4 Dropping phenomenon at different polyvinyl alcohol dosages

[0075]

[0076] In addition, if Figure 6 As shown in the data, when the PVA concentration was in the range of 6%-8%, with the increase of PVA concentration, the indole degradation rate increased accordingly and remained at 67.1%-87.3%. When the PVA content was in the range of 8%-11%, the indole degradation rate decreased with the increase of PVA content. When the PVA concentration was 11%, the indole degradation rate was 75.6%.

[0077] From the above results, it can be seen that the best PVA dosage is 8%, at which time the degradation rate of immobilized bead indole is 87.3%.

[0078] The orthogonal test was further conducted using the single factor test results to determine the optimal conditions for the immobilization of Enterococcus hirae IDO5. The orthogonal test design for the immobilized beads and its results are shown in Tables 5 and 6 below, respectively.

[0079] Table 5 Orthogonal experimental design table

[0080]

[0081] Table 6 Range analysis table of orthogonal experiment results

[0082]

[0083] By performing range analysis on the orthogonal experimental results, it can be seen from Table 6 that the order of influence of the factors affecting the preparation of immobilized beads on the indole degradation rate is: CaCl2>PVA>SA>H3BO4.

[0084] Different factors have different effects on indole degradation rate. Figure 7 (Where A is the PVA factor, B is the SA factor, C is the CaCl2 factor, and D is the H3BO4 factor) It can be seen that the best solution for immobilizing the beads is PVA=8.0%, SA=1.5%, CaCl2=3.5%, and H3BO4=3.5%.

[0085] The orthogonal test results were analyzed by variance analysis. As shown in Table 7 below, the amount of PVA and CaCl2 had a significant effect on the degradation of indole by the prepared immobilized beads (p<0.05), while the amount of PVA and H3BO4 had no significant effect on the degradation of indole by the immobilized beads (p>0.05). In the variance analysis, the order of the influence of the factors affecting the preparation of the immobilized beads on the degradation rate of indole was: CaCl2>PVA>SA>H3BO4.

[0086] Table 7 Analysis of variance table of orthogonal experiment results

[0087]

[0088] In addition, the immobilized beads prepared by the preparation method of the present invention can be specifically applied to the treatment of aquaculture manure or aquaculture wastewater.

[0089] (4) Degradation effect of Enterococcus hirae IDO5 immobilized pellets on odorous substances in pig manure wastewater:

[0090] 2 kg of Enterococcus hirae IDO5 immobilized pellets and an equal amount of Enterococcus hirae IDO5 bacterial suspension were added to two cylindrical containers containing 8 L of pig manure wastewater, and another cylindrical container containing 8 L of pig manure wastewater was used as a blank control group. After aerobic aeration treatment for 8 days, the indole and skatole contents in the pig manure wastewater were detected, and the results are shown in Table 8.

[0091] Table 8 Degradation effect of Enterococcus hirae IDO5 immobilized pellets on odorous substances in pig manure wastewater

[0092]

[0093] As shown in Table 8, after Enterococcus hirae IDO5 was prepared into immobilized pellets, the degradation effect of Enterococcus hirae IDO5 on odorous substances such as indole and skatole in pig manure wastewater was significantly improved.

[0094] Compared with the prior art, the present invention provides a preparation method and application of immobilized beads of deodorizing bacteria, which have the following beneficial effects: (1) The present invention adopts raw materials such as polyvinyl alcohol, boric acid, sodium alginate, and calcium chloride to prepare immobilized beads of Enterococcus hirae IDO5, and the required equipment is simple and the preparation process is simple; (2) The immobilized beads prepared by the present invention expand the contact area between the deodorizing microorganism Enterococcus hirae IDO5 and the odorous substances, enhance the microorganism's resistance to the influence of adverse environment, and improve the adsorption and degradation efficiency of odorous substances; (3) The immobilized beads of Enterococcus hirae IDO5 have a good deodorizing effect, with a degradation rate of indole in pig manure wastewater as high as 93.0%, and a degradation rate of skatole as high as 81.6%. When applied to livestock and poultry manure and aquaculture wastewater, it can effectively reduce the concentration of odorous substances such as indole and skatole, and can effectively reduce the odor pollution caused by livestock and poultry breeding.

[0095] It should be noted that the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0096] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing immobilized beads of deodorizing bacteria, characterized in that: The following steps are involved: S1: prepare culture medium; S2: using the culture medium prepared in step S1 to prepare an Enterococcus hirae IDO5 bacterial suspension; S3: polyvinyl alcohol, sodium alginate and distilled water are mixed, heated in a water bath to form a PVA-SA embedding agent solution, and further sterilized at high temperature and high pressure and then cooled; S4: adding the Enterococcus hirae IDO5 bacterial suspension prepared in step S2 to the PVA-SA embedding agent solution cooled in step S3, and stirring well; S5: dissolving boric acid and calcium chloride in distilled water to prepare a H3BO4-CaCl2 crosslinking agent solution; S6: taking the mixed solution prepared in step S4, adding it to the H3BO4-CaCl2 cross-linking agent solution in step S5, and obtaining the immobilized beads after cross-linking; In step S1, the culture medium is specifically LB culture medium; Step S1 specifically comprises: dissolving peptone, yeast powder and sodium chloride in distilled water, adjusting the pH of the culture medium to 7.3, and sterilizing for 20 min; Step S2 specifically includes inoculating the Enterococcus hirae IDO5 strain into the sterilized LB medium; In the immobilized beads, the mass percentage of polyvinyl alcohol is 8.0%, the mass percentage of sodium alginate is 1.5%, the mass percentage of calcium chloride is 3.5%, and the mass percentage of boric acid is 3.5%.

2. The method for preparing the immobilized deodorant bacteria pellet according to claim 1, characterized in that: In step S3, the PVA-SA embedding agent solution is sterilized under high temperature and high pressure and then cooled to room temperature.

3. The method for preparing the immobilized deodorant bacteria pellet according to claim 1, characterized in that: In step S6, the mixed solution prepared in step S4 is sucked by a 2.5 mL syringe without a needle, and is slowly added dropwise to the H3BO4-CaCl2 cross-linking agent solution in step S5 at a uniform speed.

4. The method for preparing the immobilized deodorizing bacteria pellet according to claim 3, characterized in that: In step S6, the cross-linking time of the mixed solution and the H3BO4-CaCl2 cross-linking agent solution is 2 hours.

5. The method for preparing the immobilized deodorizing bacteria pellet according to claim 1, characterized in that: After step S6, the method further includes step S7: washing the immobilized beads obtained in step S6 with distilled water and placing them in a refrigerator for later use.

6. The method for preparing the immobilized deodorant bacteria pellet according to claim 5, characterized in that: In step S7, the immobilized beads are washed three times with distilled water and placed in a refrigerator at 4°C for later use.

7. An application of an immobilized ball of deodorizing bacteria, characterized in that: The immobilized beads prepared according to any one of claims 1 to 6 are applied to the treatment of aquaculture manure or aquaculture wastewater.

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